bims-lysosi 2022-08-14 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022‒08‒14
forty papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Raptor downregulation rescues neuronal phenotypes in mouse models of Tuberous Sclerosis Complex.
mTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1.
Structures of the mannose-6-phosphate pathway enzyme, GlcNAc-1-phosphotransferase.
β-Glucocerebrosidase Deficiency Activates an Aberrant Lysosome-Plasma Membrane Axis Responsible for the Onset of Neurodegeneration.
Therapies for lysosomal storage diseases: Principles, practice, and prospects for refinements based on evolving science.
Endolysosomal cholesterol export: More than just NPC1.
MEK1/2 inhibition rescues neurodegeneration by TFEB-mediated activation of autophagic lysosomal function in a model of Alzheimer's Disease.
Harmol promotes α-synuclein degradation and improves motor impairment in Parkinson's models via regulating autophagy-lysosome pathway.
A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.
Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account.
The regulation of skin homeostasis, repair and the pathogenesis of skin diseases by spatiotemporal activation of epidermal mTOR signaling.
A Robust Nanoparticle-based Magnetic Separation Method for Intact Lysosomes.
Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.
Autolysosomal acidification failure as a primary driver of Alzheimer disease pathogenesis.
mTOR Complex 1 Content and Regulation Is Adapted to Animal Longevity.
DARPP32, a target of hyperactive mTORC1 in the retinal pigment epithelium.
The a subunit isoforms of vacuolar-type proton ATPase exhibit differential distribution in mouse perigastrulation embryos.
Elevation of gangliosides in four brain regions from Parkinson's disease patients with a GBA mutation.
FERARI and cargo adaptors coordinate cargo flow through sorting endosomes.
Two-pore channels: going with the flows.
PEBP4 Directs the Malignant Behavior of Hepatocellular Carcinoma Cells via Regulating mTORC1 and mTORC2.
mTORC1 is required for epigenetic silencing during β-cell functional maturation.
The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics.
The Deficiency of SCARB2/LIMP-2 Impairs Metabolism via Disrupted mTORC1-Dependent Mitochondrial OXPHOS.
Glial control of sphingolipid levels sculpts diurnal remodeling in a circadian circuit.
ER-Phagy: Quality and Quantity Control of the Endoplasmic Reticulum by Autophagy.
Restructured membrane contacts rewire organelles for human cytomegalovirus infection.
Regulation of TFEB nuclear localization by HSP90AA1 promotes autophagy and longevity.
The VINE complex is an endosomal VPS9-domain GEF and SNX-BAR coat.
Searching for new mTOR kinase inhibitors: Analysis of binding sites and validation of docking protocols.
Impact of 100 LRRK2 variants linked to Parkinson's Disease on kinase activity and microtubule binding.
PFN4 is required for manchette development and acrosome biogenesis during spermiogenesis.
Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2.
GOLM1 depletion modifies cellular sphingolipid metabolism and adversely affects cell growth.
Generation of TSC1 knockout induced pluripotent stem cell (iPSC) line.
Xanthomatous Giant Cell Renal Cell Carcinoma: Another Morphologic Form of TSC-associated Renal Cell Carcinoma.
Involvement of a Cluster of Basic Amino Acids in Phosphorylation-Dependent Functional Repression of the Ceramide Transport Protein CERT.
NPC1 Deficiency Contributes to Autophagy-Dependent Ferritinophagy in HEI-OC1 Auditory Cells.
Endoplasmic Reticulum Architecture and Inter-Organelle Communication in Metabolic Health and Disease.
TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation.

Nat Commun. 2022 Aug 09. 13(1): 4665

Raptor downregulation rescues neuronal phenotypes in mouse models of Tuberous Sclerosis Complex.

Vasiliki Karalis, Franklin Caval-Holme, Helen S Bateup.

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in the TSC1 or TSC2 genes, which encode proteins that negatively regulate mTOR complex 1 (mTORC1) signaling. Current treatment strategies focus on mTOR inhibition with rapamycin and its derivatives. While effective at improving some aspects of TSC, chronic rapamycin inhibits both mTORC1 and mTORC2 and is associated with systemic side-effects. It is currently unknown which mTOR complex is most relevant for TSC-related brain phenotypes. Here we used genetic strategies to selectively reduce neuronal mTORC1 or mTORC2 activity in mouse models of TSC. We find that reduction of the mTORC1 component Raptor, but not the mTORC2 component Rictor, rebalanced mTOR signaling in Tsc1 knock-out neurons. Raptor reduction was sufficient to improve several TSC-related phenotypes including neuronal hypertrophy, macrocephaly, impaired myelination, network hyperactivity, and premature mortality. Raptor downregulation represents a promising potential therapeutic intervention for the neurological manifestations of TSC.

DOI: https://doi.org/10.1038/s41467-022-31961-6
Nat Commun. 2022 Aug 10. 13(1): 4685

mTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1.

Stéphanie Kaeser-Pebernard, Christine Vionnet, Muriel Mari, Devanarayanan Siva Sankar, Zehan Hu, Carole Roubaty, Esther Martínez-Martínez, Huiyuan Zhao, Miguel Spuch-Calvar, Alke Petri-Fink, Gregor Rainer, Florian Steinberg, Fulvio Reggiori, Jörn Dengjel.

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.

DOI: https://doi.org/10.1038/s41467-022-32487-7
Proc Natl Acad Sci U S A. 2022 Aug 16. 119(33): e2203518119

Structures of the mannose-6-phosphate pathway enzyme, GlcNAc-1-phosphotransferase.

Alexei Gorelik, Katalin Illes, Khanh Huy Bui, Bhushan Nagar.

  The mannose-6-phosphate (M6P) pathway is responsible for the transport of hydrolytic enzymes to lysosomes. N-acetylglucosamine-1-phosphotransferase (GNPT) catalyzes the first step of tagging these hydrolases with M6P, which when recognized by receptors in the Golgi diverts them to lysosomes. Genetic defects in the GNPT subunits, GNPTAB and GNPTG, cause the lysosomal storage diseases mucolipidosis types II and III. To better understand its function, we determined partial three-dimensional structures of the GNPT complex. The catalytic domain contains a deep cavity for binding of uridine diphosphate-N-acetylglucosamine, and the surrounding residues point to a one-step transfer mechanism. An isolated structure of the gamma subunit of GNPT reveals that it can bind to mannose-containing glycans in different configurations, suggesting that it may play a role in directing glycans into the active site. These findings may facilitate the development of therapies for lysosomal storage diseases.

Keywords:  GlcNAc-1-phosphotransferase; lysosome; mannose-6-phosphate

DOI:  https://doi.org/10.1073/pnas.2203518119
Cells. 2022 Jul 29. pii: 2343. [Epub ahead of print]11(15):

β-Glucocerebrosidase Deficiency Activates an Aberrant Lysosome-Plasma Membrane Axis Responsible for the Onset of Neurodegeneration.

Giulia Lunghi, Emma Veronica Carsana, Nicoletta Loberto, Laura Cioccarelli, Simona Prioni, Laura Mauri, Rosaria Bassi, Stefano Duga, Letizia Straniero, Rosanna Asselta, Giulia Soldà, Alessio Di Fonzo, Emanuele Frattini, Manuela Magni, Nara Liessi, Andrea Armirotti, Elena Ferrari, Maura Samarani, Massimo Aureli.

  β-glucocerebrosidase is a lysosomal hydrolase involved in the catabolism of the sphingolipid glucosylceramide. Biallelic loss of function mutations in this enzyme are responsible for the onset of Gaucher disease, while monoallelic β-glucocerebrosidase mutations represent the first genetic risk factor for Parkinson's disease. Despite this evidence, the molecular mechanism linking the impairment in β-glucocerebrosidase activity with the onset of neurodegeneration in still unknown. In this frame, we developed two in vitro neuronal models of β-glucocerebrosidase deficiency, represented by mouse cerebellar granule neurons and human-induced pluripotent stem cells-derived dopaminergic neurons treated with the specific β-glucocerebrosidase inhibitor conduritol B epoxide. Neurons deficient for β-glucocerebrosidase activity showed a lysosomal accumulation of glucosylceramide and the onset of neuronal damage. Moreover, we found that neurons react to the lysosomal impairment by the induction of their biogenesis and exocytosis. This latter event was responsible for glucosylceramide accumulation also at the plasma membrane level, with an alteration in lipid and protein composition of specific signaling microdomains. Collectively, our data suggest that β-glucocerebrosidase loss of function impairs the lysosomal compartment, establishing a lysosome-plasma membrane axis responsible for modifications in the plasma membrane architecture and possible alterations of intracellular signaling pathways, leading to neuronal damage.

Keywords:  GBA1; Gaucher disease; glucosylceramide; lipid rafts; lysosomes; plasma membrane

DOI:  https://doi.org/10.3390/cells11152343
Mol Genet Metab. 2022 Jul 30. pii: S1096-7192(22)00381-X. [Epub ahead of print]137(1-2): 81-91

Therapies for lysosomal storage diseases: Principles, practice, and prospects for refinements based on evolving science.

Gregory A Grabowski, Pramod K Mistry.



Keywords:  Glycosphingolipidoses; Lyso-lipids; Mucopolysaccharidoses; Pompe disease; mTORC1

DOI:  https://doi.org/10.1016/j.ymgme.2022.07.014
Bioessays. 2022 Aug 07. e2200111

Endolysosomal cholesterol export: More than just NPC1.

Albert Lu.

  NPC1 plays a central role in cholesterol egress from endolysosomes, a critical step for maintaining intracellular cholesterol homeostasis. Despite recent advances in the field, the full repertoire of molecules and pathways involved in this process remains unknown. Emerging evidence suggests the existence of NPC1-independent, alternative routes. These may involve vesicular and non-vesicular mechanisms, as well as release of extracellular vesicles. Understanding the underlying molecular mechanisms that bypass NPC1 function could have important implications for the development of therapies for lysosomal storage disorders. Here we discuss how cholesterol may be exported from lysosomes in which NPC1 function is impaired.

Keywords:  NPC1; NPC1 bypass; cholesterol homeostasis; cholesterol transport; endolysosome

DOI:  https://doi.org/10.1002/bies.202200111
Mol Psychiatry. 2022 Aug 10.

MEK1/2 inhibition rescues neurodegeneration by TFEB-mediated activation of autophagic lysosomal function in a model of Alzheimer's Disease.

Yoon Sun Chun, Mi-Yeon Kim, Sun-Young Lee, Mi Jeong Kim, Tae-Joon Hong, Jae Kyong Jeon, Dulguun Ganbat, Hyoung Tae Kim, Sang Seong Kim, Tae-In Kam, Sungho Han.

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder, which is characterized by cognitive deficit due to synaptic loss and neuronal death. Extracellular amyloid β plaques are one of the pathological hallmarks of AD. The autophagic lysosomal pathway is the essential mechanism to maintain cellular homeostasis by driving clearance of protein aggregates and is dysfunctional in AD. Here, we showed that inhibiting MEK/ERK signaling using a clinically available MEK1/2 inhibitor, trametinib (GSK1120212, SNR1611), induces the protection of neurons through autophagic lysosomal activation mediated by transcription factor EB (TFEB) in a model of AD. Orally administered trametinib recovered impaired neural structures, cognitive functions, and hippocampal long-term potentiation (LTP) in 5XFAD mice. Trametinib also reduced Aβ deposition via induction of autophagic lysosomal activation. RNA-sequencing analysis revealed upregulation of autophagic lysosomal genes by trametinib administration. In addition, trametinib inhibited TFEB phosphorylation at Ser142 and promoted its nuclear translocation, which in turn induced autophagic lysosomal related genes, indicating that trametinib activates the autophagic lysosomal process through TFEB activation. From these observations, we concluded that MEK inhibition provides neuronal protection from the Aβ burden by increasing autophagic lysosomal activity. Thus, MEK inhibition may be an effective therapeutic strategy for AD.

DOI: https://doi.org/10.1038/s41380-022-01713-5
NPJ Parkinsons Dis. 2022 Aug 06. 8(1): 100

Harmol promotes α-synuclein degradation and improves motor impairment in Parkinson's models via regulating autophagy-lysosome pathway.

Jie Xu, Yun-Lin Ao, Chunhui Huang, Xiubao Song, Guiliang Zhang, Wei Cui, Yuqiang Wang, Xiao-Qi Zhang, Zaijun Zhang.

The abnormal accumulation of α-synuclein (α-syn) is a crucial factor for the onset and pathogenesis of Parkinson's disease (PD), and the autophagy-lysosome pathway (ALP) contributes to α-syn turnover. AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) regulate autophagy by initiating the macroautophagy cascade and promoting lysosomal biogenesis via increased transcription factor EB (TFEB) activity. Hence, activation of AMPK-mTOR-TFEB axis-mediated autophagy might promote α-syn clearance in PD. Harmol is a β-carboline alkaloid that has been extensively studied in a variety of diseases but rarely in PD models. In this study, we aimed to evaluate the effect and underlying mechanism of harmol in PD models in vitro and in vivo. We show that harmol reduces α-syn via ALP in a dose- and time-dependent manner in cell model that overexpressed human A53T mutant α-syn. We also demonstrate that harmol promotes the translocation of TFEB into the nucleus and accompanies the restoration of autophagic flux and lysosomal biogenesis. Importantly, harmol improves motor impairment and down-regulates α-syn levels in the substantia nigra and prefrontal cortex in the α-syn transgenic mice model. Further studies revealed that harmol might activate ALP through AMPK-mTOR-TFEB to promote α-syn clearance. These in vitro and in vivo improvements demonstrate that harmol activates the AMPK-mTOR-TFEB mediated ALP pathway, resulting in reduced α-syn, and suggesting the potential benefit of harmol in the treatment of PD.

DOI: https://doi.org/10.1038/s41531-022-00361-4
Elife. 2022 Aug 08. pii: e78923. [Epub ahead of print]11

A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.

Willa Wen-You Yim, Hayashi Yamamoto, Noboru Mizushima.

  Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the GFP-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g., GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant RFP accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g., Halo-LC3 and Halo-GFP) are proteolytically processed to generate Haloligand when delivered into lysosomes by autophagy. Hence, the amount of free Haloligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.

Keywords:  cell biology; human; mouse

DOI:  https://doi.org/10.7554/eLife.78923
Cells. 2022 Aug 02. pii: 2368. [Epub ahead of print]11(15):

Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account.

Sandip Patel, Yu Yuan, Cheng-Chang Chen, Dawid Jaślan, Gihan Gunaratne, Christian Grimm, Taufiq Rahman, Jonathan S Marchant.

  Two-pore channels TPC1 and TPC2 are ubiquitously expressed pathophysiologically relevant proteins that reside on endolysosomal vesicles. Here, we review the electrophysiology of these channels. Direct macroscopic recordings of recombinant TPCs expressed in enlarged lysosomes in mammalian cells or vacuoles in plants and yeast demonstrate gating by the Ca2+-mobilizing messenger NAADP and/or the lipid PI(3,5)P2. TPC currents are regulated by H+, Ca2+, and Mg2+ (luminal and/or cytosolic), as well as protein kinases, and they are impacted by single-nucleotide polymorphisms linked to pigmentation. Bisbenzylisoquinoline alkaloids, flavonoids, and several approved drugs demonstrably block channel activity. Endogenous TPC currents have been recorded from a number of primary cell types and cell lines. Many of the properties of endolysosomal TPCs are recapitulated upon rerouting channels to the cell surface, allowing more facile recording through conventional electrophysiological means. Single-channel analyses have provided high-resolution insight into both monovalent and divalent permeability. The discovery of small-molecule activators of TPC2 that toggle the ion selectivity from a Ca2+-permeable (NAADP-like) state to a Na+-selective (PI(3,5)P2-like) state explains discrepancies in the literature relating to the permeability of TPCs. Identification of binding proteins that confer NAADP-sensitive currents confirm that indirect, remote gating likely underpins the inconsistent observations of channel activation by NAADP.

Keywords:  NAADP; PI(3,5)P2; TPC1; TPC2; TPCN1; TPCN2; endosomes; lysosomes

DOI:  https://doi.org/10.3390/cells11152368
Front Cell Dev Biol. 2022 ;10 950973

The regulation of skin homeostasis, repair and the pathogenesis of skin diseases by spatiotemporal activation of epidermal mTOR signaling.

Juan Wang, Baiping Cui, Zhongjian Chen, Xiaolei Ding.

  The epidermis, the outmost layer of the skin, is a stratified squamous epithelium that protects the body from the external world. The epidermis and its appendages need constantly renew themselves and replace the damaged tissues caused by environmental assaults. The mechanistic target of rapamycin (mTOR) signaling is a central controller of cell growth and metabolism that plays a critical role in development, homeostasis and diseases. Recent findings suggest that mTOR signaling is activated in a spatiotemporal and context-dependent manner in the epidermis, coordinating diverse skin homeostatic processes. Dysregulation of mTOR signaling underlies the pathogenesis of skin diseases, including psoriasis and skin cancer. In this review, we discuss the role of epidermal mTOR signaling activity and function in skin, with a focus on skin barrier formation, hair regeneration, wound repair, as well as skin pathological disorders. We propose that fine-tuned control of mTOR signaling is essential for epidermal structural and functional integrity.

Keywords:  epidermis; keratinocytes; mTOR; metabolism; wound healing

DOI:  https://doi.org/10.3389/fcell.2022.950973
Bio Protoc. 2022 Jul 05. pii: e4453. [Epub ahead of print]12(13):

A Robust Nanoparticle-based Magnetic Separation Method for Intact Lysosomes.

The Son Le, Mari Takahashi, Shinya Maenosono.

  Lysosome isolation is a preresiquite for identifying lysosomal protein composition by mass spectroscopic analysis, to reveal lysosome functions, and their involvement in some diseases. Magnetic nanoparticle-based fractionation has received great attention for lysosome isolation, owing to its high efficiency, purity, and preservation of lysosomal structures. Understanding the intracellular trafficking of magnetic probes is the key point of this technique, to determine the appropriate time for magnetic isolation of lysosomes, because this parameter changes depending on different cell lines used. The traditional magnetic probes, such as superparamagnetic iron oxide nanoparticles (SPIONs), require surface modification by fluorescent dyes to enable the investigation of their intracellular trafficking, which has some disadvantages, including the possible alternation of their bio-interaction, and the instability of fluorescence properties in the lysosomal environment. To overcome those limitations, we present a protocol that employs magnetic-plasmonic nanoparticles (MPNPs) to investigate intracellular trafficking using their intrinsic imaging capability, followed by quick lysosome isolation using a magnetic column. This protocol can be easily applied to isolate the intact lysosomes of any adherent cell lines. Graphical abstract.

Keywords:   Endocytosis ; Endolysosomal pathway ; Intracellular trafficking ; Lysosomes ; Magnetic separation ; Nanoparticles ; Plasmonic imaging

DOI:  https://doi.org/10.21769/BioProtoc.4453
J Biol Chem. 2022 Aug 03. pii: S0021-9258(22)00791-8. [Epub ahead of print] 102348

Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

Mariela Nunez Santos, Daniel H Paushter, Tingting Zhang, Xiaochun Wu, Tuancheng Feng, Jiaoying Lou, Huan Du, Stephanie M Becker, Robert Fragoza, Haiyuan Yu, Fenghua Hu.

  Progranulin (PGRN) is a glycoprotein implicated in several neurodegenerative diseases. It is highly expressed in microglia and can be secreted or delivered to the lysosome compartment. PGRN comprises 7.5 granulin repeats and is processed into individual granulin peptides within the lysosome, but the functions of these peptides are largely unknown. Here, we identify CD68, a lysosome membrane protein highly expressed in microglia, as a binding partner of PGRN and PGRN-derived granulin E. Deletion analysis of CD68 showed that this interaction is mediated by the mucin-proline-rich domain of CD68. While CD68 deficiency does not affect the lysosomal localization of PGRN, it results in a specific decrease in the levels of granulin E, but no other granulin peptides. On the other hand, the deficiency of PGRN, and its derivative granulin peptides, leads to a significant shift in the molecular weight of CD68, without altering CD68 localization within the cell. Our results support that granulin E and CD68 reciprocally regulate each other's protein homeostasis.

Keywords:  CD68; Progranulin; frontotemporal dementia; granulin; lysosome; microglia

DOI:  https://doi.org/10.1016/j.jbc.2022.102348
Autophagy. 2022 Aug 10.

Autolysosomal acidification failure as a primary driver of Alzheimer disease pathogenesis.

Ju-Hyun Lee, Ralph A Nixon.

  Genetic evidence has increasingly linked lysosome dysfunction to an impaired autophagy-lysosomal pathway (ALP) flux in Alzheimer disease (AD) although the relationship of these abnormalities to other pathologies is unclear. In our recent investigation on the origin of impaired autophagic flux in AD, we established the critical early role of defective lysosomes in 5 mouse AD models. To assess in vivo alterations of autophagy and ALP vesicle acidification, we expressed eGFP-mRFP-LC3 specifically in neurons. We discovered that autophagy dysfunction in these models arises from exceptionally early failure of autolysosome/lysosome acidification, which then drives downstream AD pathogenesis. Extreme autophagic stress in compromised but still intact neurons causes AVs containing toxic APP metabolites, Aβ/β-CTFs, to pack into huge blebs and protrude from the perikaryon membrane. Most notably, AVs also coalesce with ER tubules and yield fibrillar β-amyloid within these tubules. Collectively, amyloid immunoreactivity within these intact neurons assumes the appearance of amyloid-plaques and, indeed, their eventual death transforms them into extracellular plaque lesions. Quantitative analysis confirms that neurons undergoing this transformation are the principal source of β-amyloid-plaques in APP-AD models. These findings prompt reconsideration of the conventionally accepted sequence of events in plaque formation and may help explain the inefficacy of Aβ/amyloid vaccine therapies.

Keywords:  APP-βCTF; Alzheimer’s disease; Aβ; LC3; acidification; autophagy; lysosome; neuritic plaque; perikaryal blebbing

DOI:  https://doi.org/10.1080/15548627.2022.2110729
Int J Mol Sci. 2022 Aug 06. pii: 8747. [Epub ahead of print]23(15):

mTOR Complex 1 Content and Regulation Is Adapted to Animal Longevity.

Natalia Mota-Martorell, Mariona Jové, Reinald Pamplona.

  Decreased content and activity of the mechanistic target of rapamycin (mTOR) signalling pathway, as well as the mTOR complex 1 (mTORC1) itself, are key traits for animal species and human longevity. Since mTORC1 acts as a master regulator of intracellular metabolism, it is responsible, at least in part, for the longevous phenotype. Conversely, increased content and activity of mTOR signalling and mTORC1 are hallmarks of ageing. Additionally, constitutive and aberrant activity of mTORC1 is also found in age-related diseases such as Alzheimer's disease (AD) and cancer. The downstream processes regulated through this network are diverse, and depend upon nutrient availability. Hence, multiple nutritional strategies capable of regulating mTORC1 activity and, consequently, delaying the ageing process and the development of age-related diseases, are under continuous study. Among these, the restriction of calories is still the most studied and robust intervention capable of downregulating mTOR signalling and feasible for application in the human population.

Keywords:  age-related diseases; ageing; longevity; mTORC1; metabolism

DOI:  https://doi.org/10.3390/ijms23158747
Proc Natl Acad Sci U S A. 2022 Aug 16. 119(33): e2207489119

DARPP32, a target of hyperactive mTORC1 in the retinal pigment epithelium.

Jiyang Cai, Christopher Litwin, Rui Cheng, Jian-Xing Ma, Yan Chen.

  The mechanistic target of rapamycin (mTOR) is assembled into signaling complexes of mTORC1 or mTORC2, and plays key roles in cell metabolism, stress response, and nutrient and growth factor sensing. Accumulating evidence from human and animal model studies has demonstrated a pathogenic role of hyperactive mTORC1 in age-related macular degeneration (AMD). The retinal pigment epithelium (RPE) is a primary injury site in AMD. In mouse models of RPE-specific deletion of Tuberous sclerosis 1 (Tsc1), which encodes an upstream suppressor of mTORC1, the hyperactivated mTORC1 metabolically reprogrammed the RPE and led to the degeneration of the outer retina and choroid (CH). In the current study, we use single-cell RNA sequencing (scRNA-seq) to identify an RPE mTORC1 downstream protein, dopamine- and cyclic AMP-regulated phosphoprotein of molecular weight 32,000 (DARPP-32). DARPP-32 was not found in healthy RPE but localized to drusen and basal linear deposits in human AMD eyes. In animal models, overexpressing DARPP-32 by adeno-associated virus (AAV) led to abnormal RPE structure and function. The data indicate that DARPP-32 is a previously unidentified signaling protein subjected to mTORC1 regulation and may contribute to RPE degeneration in AMD.

Keywords:  RPE; age-related macular degeneration; drusen; mTORC1

DOI:  https://doi.org/10.1073/pnas.2207489119
Sci Rep. 2022 Aug 10. 12(1): 13590

The a subunit isoforms of vacuolar-type proton ATPase exhibit differential distribution in mouse perigastrulation embryos.

Ge-Hong Sun-Wada, Yoh Wada.

Vacuolar-type H+-ATPases (V-ATPases) are large multi-subunit complexes that play critical roles in the acidification of a variety of intracellular or extracellular compartments. Mammalian cells contain four isoforms of the membrane integral subunit a (a1-a4); these isoforms contain the information necessary to target the enzyme to different cellular destinations. They are also involved in regulating the efficiency of ATP hydrolysis and proton transport. Previously, we showed that early embryogenesis requires V-ATPase function, and the luminal acidic endocytic and lysosomal compartments in the visceral endoderm of mouse embryos at the pre-gastrulation stage (E6.5) are essential for both nutrition and signal transduction during early embryogenesis. In this study, we examined the expression and distribution of a subunit isoforms in mouse embryos at E6.5. We found that all four isoforms expressed and exhibited differential distribution in the E6.5 embryo. At this developmental stage, the embryos establish highly elaborate endocytic compartments called apical vacuoles, on which the a3 isoform specifically accumulated.

DOI: https://doi.org/10.1038/s41598-022-18002-4
NPJ Parkinsons Dis. 2022 Aug 06. 8(1): 99

Elevation of gangliosides in four brain regions from Parkinson's disease patients with a GBA mutation.

Shani Blumenreich, Tamar Nehushtan, Or B Barav, Jennifer T Saville, Tamir Dingjan, John Hardy, Maria Fuller, Anthony H Futerman.

A number of genetic risk factors have been identified over the past decade for Parkinson's Disease (PD), with variants in GBA prominent among them. GBA encodes the lysosomal enzyme that degrades the glycosphingolipid, glucosylceramide (GlcCer), with the activity of this enzyme defective in Gaucher disease. Based on the ill-defined relationship between glycosphingolipid metabolism and PD, we now analyze levels of various lipids by liquid chromatography/electrospray ionization-tandem mass spectrometry in four brain regions from age- and sex-matched patient samples, including idiopathic PD, PD patients with a GBA mutation and compare both to control brains (n = 21 for each group) obtained from individuals who died from a cause unrelated to PD. Of all the glycerolipids, sterols, and (glyco)sphingolipids (251 lipids in total), the only lipid class which showed significant differences were the gangliosides (sialic acid-containing complex glycosphingolipids), which were elevated in 3 of the 4 PD-GBA brain regions. There was no clear correlation between levels of individual gangliosides and the genetic variant in Gaucher disease [9 samples of severe (neuronopathic), 4 samples of mild (non-neuronopathic) GBA variants, and 8 samples with low pathogenicity variants which have a higher risk for development of PD]. Most brain regions, i.e. occipital cortex, cingulate gyrus, and striatum, did not show a statistically significant elevation of GlcCer in PD-GBA. Only one region, the middle temporal gyrus, showed a small, but significant elevation in GlcCer concentration in PD-GBA. We conclude that changes in ganglioside, but not in GlcCer levels, may contribute to the association between PD and GBA mutations.

DOI: https://doi.org/10.1038/s41531-022-00363-2
Nat Commun. 2022 Aug 08. 13(1): 4620

FERARI and cargo adaptors coordinate cargo flow through sorting endosomes.

Jachen A Solinger, Harun-Or Rashid, Anne Spang.

Cellular organization, compartmentalization and cell-to-cell communication are crucially dependent on endosomal pathways. Sorting endosomes provide a transit point for various trafficking pathways and decide the fate of proteins: recycling, secretion or degradation. FERARI (Factors for Endosome Recycling and Rab Interactions) play a key role in shaping these compartments and coordinate Rab GTPase function with membrane fusion and fission of vesicles through a kiss-and-run mechanism. Here, we show that FERARI also mediate kiss-and-run of Rab5-positive vesicles with sorting endosomes. During these encounters, cargo flows from Rab5-positive vesicles into sorting endosomes and from there in Rab11-positive vesicles. Cargo flow from sorting endosomes into Rab11 structures relies on the cargo adaptor SNX6, while cargo retention in the Rab11 compartment is dependent on AP1. The available cargo amount appears to regulate the duration of kisses. We propose that FERARI, together with cargo adaptors, coordinate the vectorial flow of cargo through sorting endosomes.

DOI: https://doi.org/10.1038/s41467-022-32377-y
Biochem Soc Trans. 2022 Aug 12. pii: BST20220229. [Epub ahead of print]

Two-pore channels: going with the flows.

Anthony J Morgan, Lora L Martucci, Lianne C Davis, Antony Galione.

  In recent years, our understanding of the structure, mechanisms and functions of the endo-lysosomal TPC (two-pore channel) family have grown apace. Gated by the second messengers, NAADP and PI(3,5)P2, TPCs are an integral part of fundamental signal-transduction pathways, but their array and plasticity of cation conductances (Na+, Ca2+, H+) allow them to variously signal electrically, osmotically or chemically. Their relative tissue- and organelle-selective distribution, together with agonist-selective ion permeabilities provides a rich palette from which extracellular stimuli can choose. TPCs are emerging as mediators of immunity, cancer, metabolism, viral infectivity and neurodegeneration as this short review attests.

Keywords:  Ca2+; NAADP; TPC; lysosomes

DOI:  https://doi.org/10.1042/BST20220229
Int J Mol Sci. 2022 Aug 08. pii: 8798. [Epub ahead of print]23(15):

PEBP4 Directs the Malignant Behavior of Hepatocellular Carcinoma Cells via Regulating mTORC1 and mTORC2.

Qiongfeng Chen, Jingguang Jin, Wenhui Guo, Zhimin Tang, Yunfei Luo, Ying Ying, Hui Lin, Zhijun Luo.

  Phosphatidylethanolamine binding protein 4 (PEBP4) is an understudied multifunctional small protein. Previous studies have shown that the expression of PEBP4 is increased in many cancer specimens, which correlates to cancer progression. The present study explored the mechanism by which PEBP4 regulates the growth and progression of hepatocellular carcinoma cells. Thus, we showed that knockdown of PEBP4 in MHCC97H cells, where its expression was relatively high, diminished activities of serine/threonine protein kinase B (PKB, also known as Akt), mammalian target of rapamycin complex 1(mTORC1), and mTORC2, events that were not restored by insulin-like growth factor 1 (IGF-1). Conversely, overexpression of PEBP4 in MHCC97L cells with the low endogenous level yielded opposite effects. Furthermore, physical association of PEBP4 with Akt, mTORC1, and mTORC2 was observed. Interestingly, introduction of AktS473D mutant, bypassing phosphorylation by mTORC2, rescued mTORC1 activity, but without effects on mTORC2 signaling. In contrast, the effect of PEBP4 overexpression on the activity of mTORC1 but not that of mTORC2 was suppressed by MK2206, a specific inhibitor of Akt. In conjunction, PEBP4 knockdown-engendered reduction of cell proliferation, migration and invasion was partially rescued by Akt S473D while increases in these parameters induced by overexpression of PEBP4 were completely abolished by MK2206, although the expression of epithelial mesenchymal transition (EMT) markers appeared to be fully regulated by the active mutant of Akt. Finally, knockdown of PEBP4 diminished the growth of tumor and metastasis, whereas they were enhanced by overexpression of PEBP4. Altogether, our study suggests that increased expression of PEBP4 exacerbates malignant behaviors of hepatocellular cancer cells through cooperative participation of mTORC1 and mTORC2.

Keywords:  Akt; HCC; PEBP4; mTORC1; mTORC2

DOI:  https://doi.org/10.3390/ijms23158798
Mol Metab. 2022 Aug 05. pii: S2212-8778(22)00128-4. [Epub ahead of print] 101559

mTORC1 is required for epigenetic silencing during β-cell functional maturation.

Qicheng Ni, Jiajun Sun, Yichen Wang, Yanqiu Wang, Jingwen Liu, Guang Ning, Weiqing Wang, Qidi Wang.

  OBJECTIVE: The mechanistic target of rapamycin comple×1 (mTORC1) is a key molecule that links nutrients, hormones, and growth factors to cell growth/function. Our previous studies have shown that mTORC1 is required for β-cell functional maturation and identity maintenance; however, the underlying mechanism is not fully understood. This work aimed to understand the underlying epigenetic mechanisms of mTORC1 in regulating β-cell functional maturation.METHODS: We performed Microarray, MeDIP-seq and ATAC-seq analysis to explore the abnormal epigenetic regulation in 8-week-old immature βRapKO islets. Moreover, DNMT3A was overexpressed in βRapKO islets by lentivirus, and the transcriptome changes and GSIS function were analyzed.
RESULTS: We identified two major epigenetic silencing mechanisms, DNMT3A-dependent DNA methylation and PRC2-dependent H3K27me3 modification, which are responsible for functional immaturity of Raptor-deficient β-cell. Overexpression of DNMT3A partially reversed the immature transcriptome pattern and restored the impaired GSIS in Raptor-deficient β-cells. Moreover, we found that Raptor directly regulated PRC2/EED2 and H3K27me3 expression levels, as well as a group of immature genes marked with H3K27me3. Combined with ATAC-seq, MeDIP-seq and ChIP-seq, we identified β-cell immature genes with either DNA methylation and/or H3K27me3 modification.
CONCLUSION: The present study advances our understanding of the nutrient sensor mTORC1, by integrating environmental nutrient supply and epigenetic modification, i.e., DNMT3A-mediated DNA methylation and PRC2-mediated histone methylation in regulating β-cell identity and functional maturation, and therefore may impact the disease risk of type 2 diabetes.

Keywords:  Dnmt3a; Epigenetic regulation; H3K27me3; Type 2 diabetes; mTORC1; β-Cell

DOI:  https://doi.org/10.1016/j.molmet.2022.101559
J Biol Chem. 2022 Aug 03. pii: S0021-9258(22)00762-1. [Epub ahead of print] 102320

The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics.

Vincent Francis, Walaa Alshafie, Rahul Kumar, Martine Girard, Bernard Brais, Peter S McPherson.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. ARSACS is caused by mutations in the SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here using time lapse imaging and purified tubulin we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in knockout cell lines, including knockdown and knockout primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adaptor for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes.

DOI: https://doi.org/10.1016/j.jbc.2022.102320
Int J Mol Sci. 2022 Aug 03. pii: 8634. [Epub ahead of print]23(15):

The Deficiency of SCARB2/LIMP-2 Impairs Metabolism via Disrupted mTORC1-Dependent Mitochondrial OXPHOS.

Yujie Zou, Jingwen Pei, Yushu Wang, Qin Chen, Minli Sun, Lulu Kang, Xuyuan Zhang, Liguo Zhang, Xiang Gao, Zhaoyu Lin.

  Deficiency in scavenger receptor class B, member 2 (SCARB2) is related to both Gaucher disease (GD) and Parkinson's disease (PD), which are both neurodegenerative-related diseases without cure. Although both diseases lead to weight loss, which affects the quality of life and the progress of diseases, the underlying molecular mechanism is still unclear. In this study, we found that Scarb2-/- mice showed significantly reduced lipid storage in white fat tissues (WAT) compared to WT mice on a regular chow diet. However, the phenotype is independent of heat production, activity, food intake or energy absorption. Furthermore, adipocyte differentiation and cholesterol homeostasis were unaffected. We found that the impaired lipid accumulation of Adiponectin-cre; Scarb2fl/fl mice was due to the imbalance between glycolysis and oxidative phosphorylation (OXPHOS). Mechanistically, the mechanistic target of rapamycin complex 1 (mTORC1)/ eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) pathway was down-regulated in Scarb2 deficient adipocytes, leading to impaired mitochondrial respiration and enhanced glycolysis. Altogether, we reveal the role of SCARB2 in metabolism regulation besides the nervous system, which provides a theoretical basis for weight loss treatment of patients with neurodegenerative diseases.

Keywords:  LIMP-2 (SCARB2); OXPHOS; glycolysis; lysosomes; mTORC1/4E-BP1; mitochondria

DOI:  https://doi.org/10.3390/ijms23158634
Neuron. 2022 Aug 06. pii: S0896-6273(22)00661-4. [Epub ahead of print]

Glial control of sphingolipid levels sculpts diurnal remodeling in a circadian circuit.

John P Vaughen, Emma Theisen, Irma Magaly Rivas-Serna, Andrew B Berger, Prateek Kalakuntla, Ina Anreiter, Vera C Mazurak, Tamy Portillo Rodriguez, Joshua D Mast, Tom Hartl, Ethan O Perlstein, Richard J Reimer, M Thomas Clandinin, Thomas R Clandinin.

  Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. We show that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.

Keywords:  Drosophila; circadian; glia; glucocerebrosidase; lysosomes; proteostasis; sLNv; sleep; sphingolipids; structural plasticity

DOI:  https://doi.org/10.1016/j.neuron.2022.07.016
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041256. [Epub ahead of print]

ER-Phagy: Quality and Quantity Control of the Endoplasmic Reticulum by Autophagy.

Haruka Chino, Noboru Mizushima.

The endoplasmic reticulum (ER) is the largest organelle and has multiple roles in various cellular processes such as protein secretion, lipid synthesis, calcium storage, and organelle biogenesis. The quantity and quality of this organelle are controlled by the ubiquitin-proteasome system and autophagy (termed "ER-phagy"). ER-phagy is defined as the degradation of part of the ER by the vacuole or lysosomes, and there are at least two types of ER-phagy: macro-ER-phagy and micro-ER-phagy. In macro-ER-phagy, ER fragments are enclosed by autophagosomes, which is mediated by ER-phagy receptors. In micro-ER-phagy, a portion of the ER is engulfed directly by the vacuole or lysosomes. In these two pathways, some proteins in the ER lumen can be recognized selectively and subjected to ER-phagy. This review summarizes our current knowledge of ER-phagy, focusing on its membrane dynamics, molecular mechanisms, substrate specificity, and physiological significance.

DOI: https://doi.org/10.1101/cshperspect.a041256
Nat Commun. 2022 Aug 11. 13(1): 4720

Restructured membrane contacts rewire organelles for human cytomegalovirus infection.

Katelyn C Cook, Elene Tsopurashvili, Jason M Needham, Sunnie R Thompson, Ileana M Cristea.

Membrane contact sites (MCSs) link organelles to coordinate cellular functions across space and time. Although viruses remodel organelles for their replication cycles, MCSs remain largely unexplored during infections. Here, we design a targeted proteomics platform for measuring MCS proteins at all organelles simultaneously and define functional virus-driven MCS alterations by the ancient beta-herpesvirus human cytomegalovirus (HCMV). Integration with super-resolution microscopy and comparisons to herpes simplex virus (HSV-1), Influenza A, and beta-coronavirus HCoV-OC43 infections reveals time-sensitive contact regulation that allows switching anti- to pro-viral organelle functions. We uncover a stabilized mitochondria-ER encapsulation structure (MENC). As HCMV infection progresses, MENCs become the predominant mitochondria-ER contact phenotype and sequentially recruit the tethering partners VAP-B and PTPIP51, supporting virus production. However, premature ER-mitochondria tethering activates STING and interferon response, priming cells against infection. At peroxisomes, ACBD5-mediated ER contacts balance peroxisome proliferation versus membrane expansion, with ACBD5 impacting the titers of each virus tested.

DOI: https://doi.org/10.1038/s41467-022-32488-6
Autophagy. 2022 Aug 08. 1-17

Regulation of TFEB nuclear localization by HSP90AA1 promotes autophagy and longevity.

Shaosong Yang, Tiejian Nie, Hua She, Kai Tao, Fangfang Lu, Yiman Hu, Lu Huang, Lin Zhu, Dayun Feng, Dan He, Jing Qi, Thomas Kukar, Long Ma, Zixu Mao, Qian Yang.

  TFEB (transcription factor EB) regulates multiple genes involved in the process of macroautophagy/autophagy and plays a critical role in lifespan determination. However, the detailed mechanisms that regulate TFEB activity are not fully clear. In this study, we identified a role for HSP90AA1 in modulating TFEB. HSP90AA1 was phosphorylated by CDK5 at Ser 595 under basal condition. This phosphorylation inhibited HSP90AA1, disrupted its binding to TFEB, and impeded TFEB's nuclear localization and subsequent autophagy induction. Pro-autophagy signaling attenuated CDK5 activity and enhanced TFEB function in an HSP90AA1-dependent manner. Inhibition of HSP90AA1 function or decrease in its expression significantly attenuated TFEB's nuclear localization and transcriptional function following autophagy induction. HSP90AA1-mediated regulation of a TFEB ortholog was involved in the extended lifespan of Caenorhabditis elegans in the absence of its food source bacteria. Collectively, these findings reveal that this regulatory process plays an important role in modulation of TFEB, autophagy, and longevity.Abbreviations : AL: autolysosome; AP: autophagosome; ATG: autophagy related; BafA1: bafilomycin A1; CDK5: cyclin-dependent kinase 5; CDK5R1: cyclin dependent kinase 5 regulatory subunit 1; CR: calorie restriction; FUDR: 5-fluorodeoxyuridine; HSP90AA1: heat shock protein 90 alpha family class A member 1; MAP1LC3: microtubule associated protein 1 light chain 3; NB: novobiocin sodium; SQSTM1: sequestosome 1; TFEB: transcription factor EB; WT: wild type.

Keywords:  Aging; CDK5; macroautophagy; phosphorylation; transcriptional factor

DOI:  https://doi.org/10.1080/15548627.2022.2105561
Elife. 2022 Aug 08. pii: e77035. [Epub ahead of print]11

The VINE complex is an endosomal VPS9-domain GEF and SNX-BAR coat.

Shawn P Shortill, Mia S Frier, Ponthakorn Wongsangaroonsri, Michael Davey, Elizabeth Conibear.

  Membrane trafficking pathways perform important roles in establishing and maintaining the endosomal network. Retrograde protein sorting from the endosome is promoted by conserved SNX-BAR-containing coat complexes including retromer which enrich cargo at tubular microdomains and generate transport carriers. In metazoans, retromer cooperates with VARP, a conserved VPS9-domain GEF, to direct an endosomal recycling pathway. The function of the yeast VARP homolog Vrl1 has been overlooked due to an inactivating mutation found in commonly studied strains. Here, we demonstrate that Vrl1 has features of a SNX-BAR coat protein and forms an obligate complex with Vin1, the paralog of the retromer SNX-BAR protein Vps5. Unique features in the Vin1 N-terminus allow Vrl1 to distinguish it from Vps5, thereby forming a complex that we have named VINE. The VINE complex occupies endosomal tubules and redistributes a conserved mannose 6-phosphate receptor-like protein from endosomes. We also find that membrane recruitment by Vin1 is essential for Vrl1 GEF activity, suggesting that VINE is a multifunctional coat complex that regulates trafficking and signaling events at the endosome.

Keywords:  S. cerevisiae; cell biology; genetics; genomics

DOI:  https://doi.org/10.7554/eLife.77035
Chem Biol Drug Des. 2022 Aug 03.

Searching for new mTOR kinase inhibitors: Analysis of binding sites and validation of docking protocols.

Dorota Stary, Eugenie Nepovimova, Kamil Kuca, Marek Bajda.

  The mammalian target of rapamycin (mTOR) is an important biological target for development of novel anticancer drugs and potential antiageing agents. Therefore, many scientific groups search for mTOR kinase inhibitors. Herein, we present structure-based approach which could be helpful in the studies on new bioactive compounds. Method validation was preceded by structural analysis of ATP catalytic cleft and FRB domain. In silico studies allowed us to point crucial amino acid residues for ligand binding and develop optimal docking protocols. The presented methodology could be applied for design and development of potential mTOR kinase inhibitors.

Keywords:  ATP-binding site; FRB domain; docking validation; inhibitors; mTOR kinase; protein structure analysis

DOI:  https://doi.org/10.1111/cbdd.14126
Biochem J. 2022 Aug 11. pii: BCJ20220161. [Epub ahead of print]

Impact of 100 LRRK2 variants linked to Parkinson's Disease on kinase activity and microtubule binding.

Alexia F Kalogeropulou, Elena Purlyte, Francesca Tonelli, Sven M Lange, Melanie Wightman, Alan R Prescott, Shalini Padmanabhan, Esther Sammler, Dario R Alessi.

  Mutations enhancing the kinase activity of LRRK2 cause Parkinson's disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:CORdimer interface [CORB (R1728H/L)]. Most activating-variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared to a biochemical kinase assay, as only a minority of activating variants [CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)], enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppressed microtubule association in the presence of a Type I kinase inhibitor [ARM(M712V), LRR(R1320S), ROC (A1442P, K1468E, S1508R), CORA(A1589S), CORB (Y1699C, R1728H/L) and WD40(R2143M, S2350I, G2385R)]. Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion/exclusion in ongoing and future LRRK2 inhibitor clinical trials.

Keywords:  G-proteins; Parkinsons disease; leucine rich repeat kinase; signalling

DOI:  https://doi.org/10.1042/BCJ20220161
Development. 2022 Aug 11. pii: dev.200499. [Epub ahead of print]

PFN4 is required for manchette development and acrosome biogenesis during spermiogenesis.

Naila Umer, Sharang Phadke, Farhad Shakeri, Lena Arévalo, Keerthika Lohanadan, Gregor Kirfel, Marc Sylvester, Andreas Buness, Hubert Schorle.

  Profilin4 (Pfn4) is expressed during spermiogenesis and localizes to the acrosome-acroplaxome-manchette complex. Here, we generated PFN4-deficient mice, with sperm displaying severe impairment in manchette formation. Interestingly, HOOK1 staining suggests that the perinuclear ring is established, however ARL3 staining is disrupted, suggesting that lack of PFN4 does not interfere with the formation of the perinuclear ring and initial localization of HOOK1, but impedes microtubular organization of the manchette. Further, amorphous head shape and flagellar defects were detected, resulting in reduced sperm motility. Disrupted cis- and trans-Golgi networks and aberrant production of proacrosomal vesicles caused impaired acrosome biogenesis. Proteomic analysis showed, that proteins ARF3, SPECC1L and FKBP1, involved in Golgi membrane trafficking and PI3K/AKT pathway, to be higher abundant in Pfn4-/- testes. Levels of PI3K, AKT, and mTOR were elevated, while AMPK level was reduced consistent with an inhibition of autophagy. This seems to result in blockage of autophagic flux, explaining the failure in acrosome formation. IVF demonstrated that PFN4 deficient sperm is capable of fertilizing zona-free oocytes, suggesting a potential treatment in case of PFN4 related human infertility.

Keywords:  Acrosome biogenesis; Male factor infertility; Manchette; PI3K/AKT signaling; Profilin 4; Sperm biology

DOI:  https://doi.org/10.1242/dev.200499
Nat Commun. 2022 Aug 11. 13(1): 4714

Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2.

Takafumi Kato, Tsukasa Kusakizako, Chunhuan Jin, Xinyu Zhou, Ryuichi Ohgaki, LiLi Quan, Minhui Xu, Suguru Okuda, Kan Kobayashi, Keitaro Yamashita, Tomohiro Nishizawa, Yoshikatsu Kanai, Osamu Nureki.

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

DOI: https://doi.org/10.1038/s41467-022-32442-6
J Lipid Res. 2022 Aug 07. pii: S0022-2275(22)00092-X. [Epub ahead of print] 100259

GOLM1 depletion modifies cellular sphingolipid metabolism and adversely affects cell growth.

Meghana Nagaraj, Marcus Höring, Maria A Ahonen, Van Dien Nguyen, You Zhou, Helena Vihinen, Eija Jokitalo, Gerhard Liebisch, P A Nidhina Haridas, Vesa M Olkkonen.

  Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesterol esters. Further, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by EM imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.

Keywords:  Ceramide; Cholesterol ester; Dihexosylceramide; GOLPH2; GP73; Glycosphingolipid; Golgi; Hexosylceramide; Mitochondrial function; Phosphatidylethanolamine

DOI:  https://doi.org/10.1016/j.jlr.2022.100259
Stem Cell Res. 2022 Aug 05. pii: S1873-5061(22)00237-9. [Epub ahead of print]64 102888

Generation of TSC1 knockout induced pluripotent stem cell (iPSC) line.

Wei Shan, Huajun Yang, Qian Ren, Qun Wang, Guipeng An.

The TSC1 gene is a tumor suppressor gene that encodes for the growth inhibitory protein hamartin. It was founded clinically relevant to tuberous sclerosis complex (TSC) and related epilepsy. Variants in TSC1 resulted in tuberous sclerosis, focal cortical dysplasia (FCD) Type II, pulmonary lymphangioleiomyomatosis and change in everolimus sensitivity. Here, we generated induced pluripotent stem cells (iPSC) from a normal individual by electroporation of peripheral blood mononuclear cells (PBMC), and further generated TSC1-knockout human iPSC line via CRISPR/Cas9 gene editing. The resulting iPSCs had normal karyotype, free of genomically integrated epitomal plasmids, expressed pluripotency markers, and maintained trilineage differentiation potential.

DOI: https://doi.org/10.1016/j.scr.2022.102888
Am J Surg Pathol. 2022 Aug 08.

Xanthomatous Giant Cell Renal Cell Carcinoma: Another Morphologic Form of TSC-associated Renal Cell Carcinoma.

Pedram Argani, Andres Matoso, Aparna Pallavajjalla, Lisa Haley, Ming Tseh-Lin, Jessica Ng, C W Chow, Tamara Lotan, Rohit Mehra.

Over the past decade, several distinct novel renal epithelial neoplasms driven by underlying tuberous sclerosis comples (TSC)/mammalian target of rapamycin (MTOR) pathway mutations have been described. We report herein two distinctive TSC2-mutated renal cell carcinomas which do not fit any previously described entity. The two renal carcinomas occurred in young patients (ages 10 and 31 y), and were characterized by highly permeative growth within the kidney with metastases to perirenal lymph nodes. The neoplastic cells were predominantly large, multinucleated giant cells having variably eosinophilic to xanthomatous cytoplasm with basophilic stippling and frequent vacuolization. While the discohesive nature of the neoplastic cells, xanthomatous cytoplasm, immunoreactivity for histiocytic markers and minimal immunoreactivity for conventional epithelial markers raised the possibility of a histiocytic neoplasm, multifocal immunoreactivity for cytokeratin 20 helped establish their epithelial nature. Despite the aggressive growth pattern of these neoplasms and lymph node metastases, mitotic figures were rare and Ki-67 indices were low (<1%). One patient with follow-up shows no evidence of disease seven years after nephrectomy with no adjuvant therapy. Next-generation sequencing demonstrated TSC2 mutations in each case. By immunohistochemistry, downstream markers of mTOR pathway activation S6K1, 4EBP1, and glycoprotein nonmetastatic melanoma protein B were all highly expressed in these neoplasms, suggesting mTOR pathway activation as the neoplastic driver. While the cytokeratin 20 immunoreactivity and focal basophilic cytoplasmic stippling suggest a relationship to eosinophilic solid and cystic renal cell carcinoma, and cytoplasmic vacuolization suggests a relationship to eosinophilic vacuolated tumor, these neoplasms appear to be distinctive given their permeative growth patterns and predominant xanthomatous giant cell morphology. Addition of cytokeratin 20 to a panel of epithelial markers helps avoid misdiagnosis in such cases.

DOI: https://doi.org/10.1097/PAS.0000000000001940
Int J Mol Sci. 2022 Aug 02. pii: 8576. [Epub ahead of print]23(15):

Involvement of a Cluster of Basic Amino Acids in Phosphorylation-Dependent Functional Repression of the Ceramide Transport Protein CERT.

Asako Goto, Daichi Egawa, Nario Tomishige, Toshiyuki Yamaji, Kentaro Shimasaki, Keigo Kumagai, Kentaro Hanada.

  Ceramide transport protein (CERT) mediates ceramide transfer from the endoplasmic reticulum to the Golgi for sphingomyelin (SM) biosynthesis. CERT is inactivated by multiple phosphorylation at the serine-repeat motif (SRM), and mutations that impair the SRM phosphorylation are associated with a group of inherited intellectual disorders in humans. It has been suggested that the N-terminal phosphatidylinositol 4-monophosphate [PtdIns(4)P] binding domain and the C-terminal ceramide-transfer domain of CERT physically interfere with each other in the SRM phosphorylated state, thereby repressing the function of CERT; however, it remains unclear which regions in CERT are involved in the SRM phosphorylation-dependent repression of CERT. Here, we identified a previously uncharacterized cluster of lysine/arginine residues that were predicted to be located on the outer surface of a probable coiled-coil fold in CERT. Substitutions of the basic amino acids in the cluster with alanine released the SRM-dependent repression of CERT activities, i.e., the synthesis of SM, PtdIns(4)P-binding, vesicle-associated membrane protein-associated protein (VAP) binding, ceramide-transfer activity, and localization to the Golgi, although the effect on SM synthesis activity was only partially compromised by the alanine substitutions, which moderately destabilized the trimeric status of CERT. These results suggest that the basic amino acid cluster in the coiled-coil region is involved in the regulation of CERT function.

Keywords:  CERT1; endoplasmic reticulum; golgi; intragenic suppression; lipid trafficking; lipid transfer protein; phosphoinositide; sphingolipid; sphingomyelin

DOI:  https://doi.org/10.3390/ijms23158576
Front Mol Biosci. 2022 ;9 952608

NPC1 Deficiency Contributes to Autophagy-Dependent Ferritinophagy in HEI-OC1 Auditory Cells.

Lihong Liang, Hongshun Wang, Jun Yao, Qinjun Wei, Yajie Lu, Tianming Wang, Xin Cao.

  Niemann-Pick type C disease (NPCD) is a rare genetic syndrome characterized by cholesterol accumulation in multiple organelles. NPCD is mainly caused by gene deficiency of NPC intracellular cholesterol transporter 1 (NPC1). It has been reported that some of the NPCD patients exhibit clinical features of progressive hearing loss at high frequency and iron disorder, but the underlying relationship is unknown. A recent study has reported that ferroptosis contributes to the impairment of cochlear hair cells that are related to sensory hearing. In this study, we generated NPC1-deficient HEI-OC1 cells to show the effect of NPC1 deficiency on cochlear outer hair cells. We found that NPC1 deficiency enhances autophagy-dependent ferritinophagy to release Fe (II). Our work provides important insights into the effect of NPC1 deficiency in auditory cells, indicating that it induces ferroptosis and results in hearing loss.

Keywords:  NCOA4; NPC1 deficiency; Niemann–Pick type C disease; ferroptosis; hearing loss

DOI:  https://doi.org/10.3389/fmolb.2022.952608
Cold Spring Harb Perspect Biol. 2022 Aug 08. pii: a041261. [Epub ahead of print]

Endoplasmic Reticulum Architecture and Inter-Organelle Communication in Metabolic Health and Disease.

Ana Paula Arruda, Güneş Parlakgül.

The endoplasmic reticulum (ER) is a key organelle involved in the regulation of lipid and glucose metabolism, proteostasis, Ca2+ signaling, and detoxification. The structural organization of the ER is very dynamic and complex, with distinct subdomains such as the nuclear envelope and the peripheral ER organized into ER sheets and tubules. ER also forms physical contact sites with all other cellular organelles and with the plasma membrane. Both form and function of the ER are highly adaptive, with a potent capacity to respond to transient changes in environmental cues such as nutritional fluctuations. However, under obesity-induced chronic stress, the ER fails to adapt, leading to ER dysfunction and the development of metabolic pathologies such as insulin resistance and fatty liver disease. Here, we discuss how the remodeling of ER structure and contact sites with other organelles results in diversification of metabolic function and how perturbations to this structural flexibility by chronic overnutrition contribute to ER dysfunction and metabolic pathologies in obesity.

DOI: https://doi.org/10.1101/cshperspect.a041261
Mol Cell. 2022 Aug 10. pii: S1097-2765(22)00663-3. [Epub ahead of print]

TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation.

Linhan Wang, Jiqiang Li, Qingchen Wang, Man-Xi Ge, Jia Ji, Di Liu, Zhiyuan Wang, Yang Cao, Yaoyang Zhang, Zai-Rong Zhang.

  Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.

Keywords:  ERAD; TMUB1; endoplasmic-reticulum-associated degradation; escortase; membrane protein extraction; membrane protein quality control; p97/VCP; protein degradation; retrotranslocation

DOI:  https://doi.org/10.1016/j.molcel.2022.07.006